Kirsten C. Sadler

Conservation and divergence of methylation patterning in plants and animals

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.

A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.

Hepatomegaly is a sign of many liver disorders. To identify zebrafish mutants to serve as models for hepatic pathologies, we screened for hepatomegaly at day 5 of embryogenesis in 297 zebrafish lines bearing mutations in genes that are essential for embryonic development. Seven mutants were identified, and three have phenotypes resembling different liver diseases. Mutation of the class C vacuolar protein sorting gene vps18 results in hepatomegaly associated with large, vesicle-filled hepatocytes, which we attribute to the failure of endosomal-lysosomal trafficking. Additionally, these mutants develop defects in the bile canaliculi and have marked biliary paucity, suggesting that vps18 also functions to traffic vesicles to the hepatocyte apical membrane and may play a role in the development of the intrahepatic biliary tree. Similar findings have been reported for individuals with arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, which is due to mutation of another class C vps gene. A second mutant, resulting from disruption of the tumor suppressor gene nf2, develops extrahepatic choledochal cysts in the common bile duct, suggesting that this gene regulates division of biliary cells during development and that nf2 may play a role in the hyperplastic tendencies observed in biliary cells in individuals with choledochal cysts. The third mutant is in the novel gene foie gras, which develops large, lipid-filled hepatocytes, resembling those in individuals with fatty liver disease. These mutants illustrate the utility of zebrafish as a model for studying liver development and disease, and provide valuable tools for investigating the molecular pathogenesis of congenital biliary disorders and fatty liver disease.

Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation.

Steatosis is the most common consequence of acute alcohol abuse and may predispose to more severe hepatic disease. Increased lipogenesis driven by the sterol response element binding protein (SREBP) transcription factors is essential for steatosis associated with chronic alcohol ingestion, but the mechanisms underlying steatosis following acute alcohol exposure are unknown. Zebrafish larvae represent an attractive vertebrate model for studying alcoholic liver disease (ALD), because they possess the pathways to metabolize alcohol, the liver is mature by 4 days post‐fertilization (dpf), and alcohol can be simply added to their water. Exposing 4 dpf zebrafish larvae to 2% ethanol (EtOH) for 32 hours achieves ∼80 mM intracellular EtOH and up‐regulation of hepatic cyp2e1, sod, and bip, indicating that EtOH is metabolized and provokes oxidant stress. EtOH‐treated larvae develop hepatomegaly and steatosis accompanied by changes in the expression of genes required for hepatic lipid metabolism. Based on the importance of SREBPs in chronic ALD, we explored the role of Srebps in this model of acute ALD. Srebp activation was prevented in gonzo larvae, which harbor a mutation in the membrane‐bound transcription factor protease 1 (mbtps1) gene, and in embryos injected with a morpholino to knock down Srebp cleavage activating protein (scap). Both gonzo mutants and scap morphants were resistant to steatosis in response to 2% EtOH, and the expression of many Srebp target genes are down‐regulated in gonzo mutant livers. Conclusion: Zebrafish larvae develop signs of acute ALD, including steatosis. Srebp activation is required for steatosis in this model. The tractability of zebrafish genetics provides a valuable tool for dissecting the molecular pathogenesis of acute ALD. (HEPATOLOGY 2008.)

UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1.

In contrast to the deregulated hepatocellular division that is a feature of many hepatic diseases and malignancies, physiologic liver growth during embryonic development and after partial hepatectomy (PH) in adults is characterized by tightly controlled cell proliferation. We used forward genetic screening in zebrafish to test the hypothesis that a similar genetic program governs physiologic liver growth during hepatogenesis and regeneration after PH. We identified the uhrf1 gene, a cell cycle regulator and transcriptional activator of top2a expression, as required for hepatic outgrowth and embryonic survival. By developing a methodology to perform PH on adult zebrafish, we found that liver regeneration inuhrf1+/− adult animals is impaired.uhrf1 transcript levels dramatically increase after PH in both mice, and zebrafish and top2a is not up-regulated in uhrf1+/− livers after PH. This indicates that uhrf1 is required for physiologic liver growth in both embryos and adults and illustrates that zebrafish livers regenerate.

New school in liver development: Lessons from zebrafish†

There is significant overlap in the genes and pathways that control liver development and those that regulate liver regeneration, hepatic progenitor cell expansion, response to injury, and cancer. Additionally, defects in liver development may underlie some congenital and perinatal liver diseases. Thus, studying hepatogenesis is important for understanding not only how the liver forms, but also how it functions. Elegant work in mice has uncovered a host of transcription factors and signaling molecules that govern the early steps of hepatic specification; however, the inherent difficulty of studying embryogenesis in utero has driven developmental biologists to seek new systems. The rapidly developing vertebrate zebrafish is a favorite model for embryology. The power of forward genetic screens combined with live real‐time imaging of development in transparent zebrafish embryos has highlighted conserved processes essential for hepatogenesis and has uncovered some exciting new players. This review presents the advantages of zebrafish for studying liver development, underscoring how studies in zebrafish and mice complement each other. In addition to their value for studying development, zebrafish models of hepatic and biliary diseases are expanding, and using these small, inexpensive embryos for drug screening has become de rigueur. Zebrafish provide a shared platform for developmental biology and translational research, offering innovative methods for studying liver development and disease. The story of hepatogenesis has something for everyone. It involves transcriptional regulation, cell‐cell interaction, signaling pathways, control of cell proliferation and apoptosis, plus morphogenic processes that sculpt vasculature, parenchymal cells, and mesenchyme to form the multifaceted liver. Decades of research on liver development in mice and other vertebrates offer valuable lessons in how the multipotent endoderm is programmed to form a functional liver. Of equal importance are insights that have illuminated the mechanisms by which hepatic progenitors are activated in a damaged liver, how the adult liver regenerates, and, possibly, the basis for engineering liver cells in vitro for cell transplantation to sustain patients with liver failure. Moreover, processes that are key to liver development are often co‐opted during pathogenesis. Therefore, reviewing hepatogenesis is informative for both basic and translational researchers. In this review, we bring to light the many advantages offered by the tropical freshwater vertebrate zebrafish (Danio rerio) in studying hepatogenesis. By comparing zebrafish and mice, we highlight how work in each system complements the other and emphasize novel paradigms that have been uncovered using zebrafish. Finally, we highlight exciting efforts using zebrafish to model hepatobiliary diseases. (HEPATOLOGY 2009.)

Endothelial Signals Modulate Hepatocyte Apicobasal Polarization in Zebrafish

Emerging evidence indicates that paracrine signals from endothelial cells play a role in tissue differentiation and organ formation [1-3]. Here, we identify a novel role for endothelial cells in modulating hepatocyte polarization during liver organogenesis. We find that in zebrafish, the apical domain of the hepatocytes predicts the location of the intrahepatic biliary network. The refinement of hepatocyte polarization coincides with the invasion of endothelial cells into the liver, and these endothelial cells migrate along the maturing basal surface of the hepatocytes. Using genetic, pharmacological, and transplantation experiments, we provide evidence that endothelial cells influence the polarization of the adjacent hepatocytes. This influence of endothelial cells on hepatocytes is mediated at least in part by the cell-surface protein Heart of glass and contributes to the establishment of coordinately aligned hepatocyte apical membranes and evenly spaced intrahepatic conduits.

Activating transcription factor 6 plays protective and pathological roles in steatosis due to endoplasmic reticulum stress in zebrafish

Many etiologies of fatty liver disease (FLD) are associated with the hyperactivation of one of the three pathways composing the unfolded protein response (UPR), which is a harbinger of endoplasmic reticulum (ER) stress. The UPR is mediated by pathways initiated by PRKR‐like endoplasmic reticulum kinase, inositol‐requiring 1A/X box binding protein 1, and activating transcription factor 6 (ATF6), and each of these pathways has been implicated to have a protective or pathological role in FLD. We used zebrafish with FLD and hepatic ER stress to explore the relationship between Atf6 and steatosis. A mutation of the foie gras (foigr) gene caused FLD and hepatic ER stress. The prolonged treatment of wild‐type larvae with tunicamycin (TN), which caused chronic ER stress, phenocopied foigr. In contrast, acute exposure to a high dose of TN robustly activated the UPR but was less effective at inducing steatosis. The sterol regulatory element binding protein transcription factors were not required for steatosis in any of these models. Instead, depleting larvae of active Atf6 either through a membrane‐bound transcription factor peptidase site 1 mutation or an atf6 morpholino injection protected them against steatosis caused by chronic ER stress, but exacerbated steatosis caused by acute TN treatment. Conclusion: ER stress causes FLD. A loss of Atf6 prevents steatosis caused by chronic ER stress but can also potentiate steatosis caused by acute ER stress. This demonstrates that Atf6 can play both protective and pathological roles in FLD. (HEPATOLOGY 2011;)

White adipose tissue development in zebrafish is regulated by both developmental time and fish size

Adipocytes are heterogeneous. Whether their differences are attributed to anatomical location or to different developmental origins is unknown. We investigated whether development of different white adipose tissue (WAT) depots in zebrafish occurs simultaneously or whether adipogenesis is influenced by the metabolic demands of growing fish. Like mammals, zebrafish adipocyte morphology is distinctive and adipocytes express cell‐specific markers. All adults contain WAT in pancreatic, subcutaneous, visceral, esophageal, mandibular, cranial, and tail‐fin depots. Unlike most zebrafish organs that form during embryogenesis, WAT was not found in embryos or young larvae. Instead, WAT was first identified in the pancreas on 12 days postfertilization (dpf), and then in visceral, subcutaneous, and cranial stores in older fish. All 30 dpf fish exceeding 10.6 mm standard length contained the adult repertoire of WAT depots. Pancreatic, esophageal, and subcutaneous WAT appearance correlated with size, not age, as found for other features appearing during postembryonic zebrafish development. Developmental Dynamics 239:3013–3023, 2010.

UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis

UHRF1 [ubiquitin-like protein, containing PHD (plant homeodomain) and RING finger domains 1] is required for cell cycle progression and epigenetic regulation. In the present study, we show that depleting cancer cells of UHRF1 causes activation of the DNA damage response pathway, cell cycle arrest in G2/M-phase and apoptosis dependent on caspase 8. The DNA damage response in cells depleted of UHRF1 is illustrated by: phosphorylation of histone H2AX on Ser139, phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr15. Moreover, we find that UHRF1 accumulates at sites of DNA damage suggesting that the cell cycle block in UHRF1-depleted cells is due to an important role in damage repair. The consequence of UHRF1 depletion is apoptosis; cells undergo activation of caspases 8 and 3, and depletion of caspase 8 prevents cell death induced by UHRF1 knockdown. Interestingly, the cell cycle block and apoptosis occurs in p53-containing and -deficient cells. From the present study we conclude that UHRF1 links epigenetic regulation with DNA replication.